Telomerase is a large ribonucleoprotein complex responsible for replicating the G-rich strand of telomeres, the physical ends of chromosomes. It is critical for maintaining telomere length and preventing chromosome instability, and is a critica determinant of cellular aging, stem cell renewal, and tumorigenesis. Telomerase activity is low or undetectable in most somatic cells, while telomerase activation is important for the immortal phenotype of most cancer cells. Despite the importance of telomerase in human health and longevity, a detailed understanding of the structural biology of telomerase is still lacking. Telomerase contains two components required for catalytic activity, a unique eukaryotic reverse transcriptase (telomerase reverse transcriptase, TERT) and a large RNA (telomerase RNA, TER) that includes a template sequence used for copying the telomere repeat, plus other species-specific proteins required for function in vivo. To address the molecular basis of telomerase catalytic activity, in this grant application we are proposing to: (1) Determine a high resolution cryoelectron microscopy (cryoEM) structure of telomerase from the model organism Tetrahymena to provide a detailed view of the TERT-TER interactions in the catalytic core; (2) Use the information from the cryoEM structure to model the catalytic core of human telomerase and investigate the functional importance of TER and TERT domain interactions observed in cryoEM maps using NMR, some X- ray crystallography, and biochemistry; (3) Determine the pathway of the telomeric DNA and TER on telomerase during the catalytic cycle using cryoEM and biochemical methods; and (4) Develop stable cell lines for purifying human telomerase for structural studies to determine the subunit organization. These experiments will reveal the structure of the catalytic core of telomerase and provide insight into the roles of TERT and TER in catalysis and the catalytic mechanism. The results of these investigations will provide fundamental insights into the contributions of domains of TERT and TER to activity, an understanding of the molecular basis for human telomerase activity, insights into how TERT and TER mutations linked to disease affect activity, and a basis for structure based design of drugs that target the catalytic core of telomerase.